Abstract
In the development of therapeutic antibodies and biosimilars, an appropriate biopharmaceutical CMC control strategy that connects critical quality attributes with mechanism of action should enable product assessment at an early stage of development in order to mitigate risk. Here we demonstrate a new analytical workflow using trastuzumab which comprises "middle-up" analysis using a combination of IdeS and the endoglycosidases EndoS and EndoS2 to comprehensively map the glycan content. Enzymatic cleavage between the two N-acetyl glucosamine residues of the chitobiose core of N-glycans significantly simplifies the oligosaccharide component enabling facile distinction of GlcNAc from GlcNAc with core fucose. This approach facilitates quantitative determination of total Fc-glycan core-afucosylation, which was in turn correlated with receptor binding affinity by surface plasmon resonance and in vitro ADCC potency with a cell based bioassay. The strategy also quantifies Fc-glycan occupancy and the relative contribution from high mannose glycans.
Highlights
A s therapeutic proteins are developed and manufactured, the U.S Food and Drug Administration and European
Medicines Agency have expectations based on a Quality by Design (QbD) initiative and, in terms of biosimilars, an examination of a Totality of Evidence.[1−3] Knowledge of the drug’s mechanism of action (MOA) and how manufacturing processes affect structural attributes and in turn function are required
With respect to recombinant monoclonal antibody products and associated biosimilars, glycosylation of the conserved Fc asparagine residue is a modification commonly recognized to be a factor in terms of product stability, structural conformation, and functional activity.[6−8] The distribution of the attached Fc-glycan population of therapeutic antibodies is considered an important critical quality attributes (CQAs), attracting particular attention due to its influence on effector functions.[8−12]
Summary
A s therapeutic proteins are developed and manufactured, the U.S Food and Drug Administration and European. A majority of the existing therapeutic antibody products are expressed in Chinese hamster ovary (CHO) cells or mouse myeloma (NS0), SP2/0 plasma cell lines.[9,16] The resulting Fcglycans (N-glycans) are typically a heterogeneous population of high mannose type and, more predominantly, biantenary complex oligosaccharides. They comprise a trimannosyl core structure, mainly with a fucose residue attached to the core N-. The relative abundance of afucosylated Fc-glycan variants detected in these recombinant monoclonal antibodies is typically low (
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
